The invention concerns a method for high gradient magnetic separation (HGMS) technology for the separation and purification of biological material.
The separation and purification of certain particles from heterogeneous particle suspensions is of great importance for a variety of analytic methods especially in the field of biomedical research. Generally, particles to be purified, called “target particles” in the following, frequently differ only minimally from the rest of the particles contained in the suspension, called “non-target particles” in the following. Target particles and non-target particles are often cells or cell fragments, however, they can be any other biological substances.
Some established separation methods use magnetic properties of target particles, where the target particles have naturally occurring “intrinsic” magnetic properties, or where the target particles are labeled through targeted attachment of synthetic magnetic particles before the actual separation procedure
Intrinsically magnetic particles, for example, are red blood cells, given that the hemoglobin contained in them exists in the de-oxygenated or oxidized, but not in the oxygenated state. Here, de-oxygenated means not oxygen carrying and oxygenated means oxygen carrying. In the latter case, the hemoglobin molecule carries an oxygen molecule with non-covalent binding (i.e., reversible binding). The oxidized state of hemoglobin has to be differentiated, in which the oxygen atoms or other oxidizing atoms are bound covalently (i.e., non-reversibly) to the central iron atom of the hemoglobin molecule.
Now the orbitals of the central iron atom contained in the hemoglobin molecule carry unpaired electrons both in the de-oxygenated as well as the oxidized form (but not in the oxygenated form). The unpaired spin of these electrons enables the induction of magnetic poles in the iron atom by application of a magnetic field.
A magnetic field of the conventional kind, however, does not exert a directed net magnetic force on a particle which contains such iron atoms, since, due to the very small atomic diameter, attracting and repelling forces at the North Pole and South Pole of the polarized iron atoms keep the balance. Also, the particle will lose its polarization after removal of the magnetic field. This kind of magnetism is known as “paramagnetism.” A special form of paramagnetism is sometimes called “superparamagnetism.” Between both these forms, however, physically no clear separation exists, therefore, in the following, the terms “paramagnetism” and “paramagnetic” shall encompass the terms “superparamagnetism” and “superparamagnetic.”
Also, the well-established synthetic secondary particles used for magnetic labeling of target particles are paramagnetic and usually contain, very similar to oxidized hemoglobin, small amounts of oxidized iron or another magnetizable substance. Moreover, they are ideally so small that they form stable colloids in suspension, in other words, they do not sediment over long periods of time (months to years); therefore, their diameter in general measures 30-200 nm. Commercially available particles of this kind are distributed alone or conjugated to antibodies, for example by chemicell GmbH (Eresburgstrasse 22-23, D-12103 Berlin, Germany), micromod Partikeltechnologie GmbH (Friedrich-Barnewitz-Str.4, D-18119 Rostock, Germany), or Miltenyi Biotech GmbH (Friedrich Ebert Straγe 68, D-51429 Bergisch Gladbach, Germany).
An established method to purify paramagnetic particles, in other words, to separate paramagnetic particles from particle suspensions, is the creation of extremely high magnetic field gradients. A sufficiently high magnetic gradient leads to the fact that north and south poles of the paramagnetic particles experience a difference in attracting and repelling forces and therefore, a directed net-magnetic force. This technology is known under the term high gradient magnetic separation (HGMS).
Here a distinction is to be made between so-called “internal” and “external” high gradient magnetic separators. First descriptions of the HGMS-technology referred to internal separators. These can be found in Oberteuffer (IEEE Transactions on Magnetics, Mag-9, No. 3, September 1973:303-306) and in the U.S. Pat. No. 3,676,337. A ferromagnetic material in a suitable, non-magnetic container which serves as separation chamber, is introduced into a strong homogenous magnetic field which can be generated by an electromagnet or by a horseshoe shaped permanent magnet (dipole magnet). In this case, the ferromagnetic material generally carries the name “matrix;” it can be of filamentary (wire-like or thread-like), of spherical (ball-like) or of an otherwise different shape, for example it can consist of punched sheet steel. The ferromagnetic material of the matrix experiences a magnetization corresponding to its magnetic susceptibility X through the externally applied field. Commencing from the surface of the matrix material, magnetic field gradients are created which can reach over 100 Tesla per centimeter; whereby the magnitude of the gradient is in the inverse ratio to the diameter of the utilized filamentary or spherical elements. “External” high gradient magnetic separators reach similarly high gradients through a technically more complex, special arrangement of the magnets outside the actual separation chamber, as for example disclosed in WO 98/055236, WO 99/019071 or U.S. Pat. No. 6,241,894 B1. As a fundamental difference, the need for a matrix inside the separation chamber does not arise here.
Nowadays, internal high gradient magnetic separators are the most widely used in biomedical research. U.S. Pat. Nos. 4,664,796 and 5,200,084 describe particular embodiments of such separators.
U.S. Pat. No. 5,200,084 discloses an apparatus and a method that is particularly directed at the purification of small amounts of biological material in the wells of a microtiter plate. Among others, purifications of up to 83% of CD4 cells labeled with paramagnetic secondary particles from peripheral mononuclear blood cells (PBMCs) are reported.
To the inventor's knowledge, at present only one technical design of an internal high gradient magnetic separator achieves high purification rates, as disclosed in the WO 96/26782 and the EP 0942766 B1. To avoid unspecific binding of non-target particles to the matrix, which impair the purification result in the named documents, the separation chamber of said separator contains a polymer-coated matrix. The polymer is drawn onto the matrix in several steps, as described in detail in the WO 96/26782. According to the information given by the authors, the so assembled separation chamber distinguishes itself by the fact that the polymer creates a hard, closed, liquid impermeable and ion impermeable coating containing less than 1% water on the ferromagnetic material of the matrix; and that the polymer-coated matrix fills 60-70% of the total volume of the described separation chamber.
According to the named publication, apart from decreasing unspecific binding of non-target particles to the matrix, said coating is supposed to also avoid damage of the biological material to be separated by direct contact with the ferromagnetic matrix material (e.g., physical damage), as well as to exclude a chemical reaction of the ferromagnetic matrix material with the buffer solution used for suspension of the biological material, since freed ions could also lead to damage of the biological material to be separated (e.g., chemical damage). Both of these kinds of damage, however, are to be considered hypothetical, since no scientifically secured findings are available. Moreover, Paul et al. conversely reported no impairment of morphology and viability of blood cells and blood cell fragments that passed a non-treated stainless steel matrix of a HGMS-column (Paul et al. Clinical and Laboratory Haematology, 7, 1985:43-53.
All patents and publications mentioned up to this point are included herein by reference.
The kind of separation chambers described are costly due to their time intensive production process particularly for the coating of the matrix. They are commercially available from Miltenyi Biotech GmbH (loc.cit.).
They achieve high purifications especially when used for particles labeled with synthetic paramagnetic particles from particle suspensions.
The application of said separation chambers with a coated matrix for intrinsically (naturally) paramagnetic particles was investigated. Here, malaria-infected red blood cells served as intrinsically paramagnetic particles. The pathogens of malaria, parasites of the genus Plasmodium from the group of the protozoa, selectively attack red blood cells and have the property to oxidize the central iron atom of the free heme molecule arising in the infected red blood cell into trivalent iron. This is, as described above, paramagnetic. Therefore, malaria infected red blood cells should be separable from non-infected and oxygenized red blood cells in separation chambers of high gradient magnetic separators. This was shown first in 1981 by Paul et al. (Lancet, Jul. 11, 1981:70-71) in 1981. At present, the described, commercially available separation chambers with coated matrix are said to achieve purifications of over 80% (Uhlemann et al., MACS&more 2000; 4 (2):7-8, Trang et al., Malaria Journal 2004; 3:1-7).
The specified investigations, however, refer to only one of the total four known pathogenic malaria pathogens in humans, namely Plasmodium falciparum, the pathogenic agent of Malaria tropica, as well as one other malaria pathogen in rodents, Plasmodium berghei. Further scientific studies about the total nearly 120 further known Plasmodium species are not available. It is known to the inventors, however, that the application of the commercially available separation chambers to the purification of red blood cells infected with Plasmodium vivax, the pathogen of Malaria tertiana also occurring in humans, does not always lead to satisfactory results.
From the forgoing explanations it is obvious that further improvements of the purification effectiveness of the HGMS technology would be of great usefulness for the field of biomedical research. This also applies with reference to the cost efficiency, since the known sophisticated separation chambers are not always available in different areas simply for cost considerations, particularly, in the investigation of malaria pathogens, which particularly affects countries with smaller medical and research budgets. Therefore, it is the task of the invention to provide a HGMS separation column that achieves better purification results in a cost-efficient way.